The goal of this project is to establish human cytomegalovirus (HCMV) as a new human vaccine vector platform. In non-human primate models, CMV-vectored vaccines have shown unprecedented protection against immunodeficiency viruses and similar vaccines are currently being evaluated for their protective potential against a number of infectious diseases and cancer. CMV-vectored vaccines represent a paradigm shift in vaccinology due to a) continuous antigen production and thus immune stimulation, b) their ability to overcome vector-specific immunity which enables repeated use of the same vector, c) the finding that spread- deficient vectors maintain long-term immune stimulation. A major barrier to translate the pre-clinical results into a vaccine for human use is the fact that all pre-clinical studies were performed using animal CMVs that do not infect humans. Since HCMV-based vectors have never been tested in humans the optimal characteristics of a human vector are unknown and an appropriate HCMV-vector backbone has yet to be selected and designed. Unfortunately, all currently available recombinant HCMV clones display genetic lesions and/or tissue culture adaptations that are expected to limit their usefulness as vector platform. Moreover, most of these clones were obtained from primary isolates of symptomatic and/or immune-suppressed individuals raising the possibility of increased pathogenicity and adaptation to an immunosuppressed environment. To overcome these limitations and to establish a clear path to regulatory approval of a new HCMV-based vector platform we propose to establish a panel of novel HCMV isolates from asymptomatic carriers, isolate and characterize molecular clones by bacterial artificial chromosome technology as well as genome sequencing. We will further introduce specific genomic modifications analogous to those observed in animal CMVs to increase immunogenicity and safety. The resulting vectors will be prioritized according to their stability during in vitro culture, viral yeld, and ability to infect different cell types. Upon completion of this project we will have a well-documented HCMV clones with optimal in vitro growth characteristics as a backbone for designing safe vaccines for HIV and other infectious diseases. PUBLIC HEALTH RELEVANCE: The goal of this project is to isolate and characterize molecular clones of human cytomegalovirus. After inserting antigens from other pathogens this clone will then be used as a new vaccine against these pathogens.